Marking apparatus for nucleic acid marking of items

ABSTRACT

A marking apparatus ( 1 ) for marking an item ( 12 ). The apparatus comprises: means to receive the item; a nucleic acid marker, means to release a marking fluid ( 8 ); and a distribution mechanism ( 11 ) coupled to the nucleic acid marker and the means to release the marking fluid. The means to release the marking fluid can be activated to release the marking fluid such that the distribution mechanism disperses a mixture of the nucleic acid marker and the marking fluid onto the item.

The present invention relates to a marking apparatus and also to amethod of marking an item and a method of analysing a marked item.

In the field of security, it is often necessary to transport largequantities of bank notes securely between locations. For example, it iscommon to transport bank notes from a bank vault to an automated tellermachine (ATM) situated in a supermarket or shopping centre where theycan be dispensed. Although the bank notes are guarded whilst in transit,they are inherently vulnerable to theft in view of the need to transportthem in civilian vehicles, especially during the time between theunloading of the vehicle and the deposit of the bank notes in the ATM.This attracts thieves due to the large amount of money that is involved.

Typically, when money is transported to an ATM, it is locked inside acassette before its departure and the entire cassette is loaded into theATM, together with the bank notes. This avoids the need for directhandling of the bank notes, which can be a security risk. Thus any thiefattempting to steal the bank notes must be able to penetrate thecassette in order to remove the bank notes and use them.

It is known in the art to defend the bank note cassettes in a number ofways. It is, of course, possible to provide a physical locking mechanismon the cassette, which helps to prevent it from being broken open.However, the problem with this approach is that a thief can steal theentire cassette, with the bank notes inside, and then open the cassettein a secret location where he has appropriate cutting tools in order toretrieve the bank notes. Thus, given sufficient time, a thief will beable to open most cassettes, despite a physical locking mechanism beingin place.

It is also known in the art to supplement the physical locking mechanismof a cassette with a device which renders the bank notes unusable upontampering with the cassette. Typically, such a device comprises areservoir of ink and a source of pressurised gas. When tampering withthe cassette is detected, the pressurised gas is released and used todisperse the ink across the bank notes held in the cassette (see, forexample, WO-A-98/03758). In such arrangements, if a thief is able tosteal a cassette containing bank notes, he will, on attempting to openthe cassette, trigger the release of the ink which indelibly colourseach of the bank notes in a clearly visible manner. The ink cannot beremoved from the bank note by washing etc. without also destroying thebank note itself. This makes the subsequent use of the bank notesimpossible because shops and banks will refuse to accept bank noteswhich have been stained in this way. Accordingly, while these devices donot prevent the theft of bank notes per se, they render such theftunprofitable because the bank notes cannot be used.

Despite the widespread use of such bank note staining devices in thecassettes used to transfer bank notes, there is still a significantproblem with the theft of bank note cassettes. On average, there areseveral such thefts each day in the UK, alone. It is commonly the casethat the police will recover large quantities of stained bank notesfollowing such thefts, the bank notes having been dumped by thieves whorealise that they will be unable to use them. However, the bank notesare not necessarily valueless to their original owner since, if he canbe identified, central banks will usually reimburse the value of banknotes that have been indelibly stained and recovered. The problem isthat there are so many thefts of bank notes that it is often difficultto determine exactly which consignment of bank notes has been recoveredat any particular time and thus who the rightful owner of the bank notesis. Thus it is difficult to ascertain who the rightful owner of the banknotes is. Without this information, reimbursement cannot take place.

This is particularly a problem in the countries of the so-called“Euro-zone” in which bank notes are equally usable in any of the membercountries. Therefore the large number of bank notes in circulation makesit even more difficult to determine in which particular theft anyrecovered bank notes were stolen.

The problems associated with the transport of bank notes have beenexemplified above in relation to the supply of money to ATMs. However,the problems also arise in the transfer of bank notes in othersituations such as between bank vaults and when bank notes are carriedby an individual in a brief case or the like.

The present invention seeks to alleviate one or more of the aboveproblems.

According to a first aspect of the present invention, there is provideda marking apparatus for marking an item, the apparatus comprising: anucleic acid marker; means to release a marking fluid; and adistribution mechanism coupled to the nucleic acid marker and the meansto release the marking fluid, the means to release the marking fluidbeing activatable to release the marking fluid such that thedistribution mechanism disperses a mixture of the nucleic acid markerand the marking fluid onto the item.

Preferably, the apparatus further comprises means to receive the item.

Conveniently the item is one or more bank notes, the means to releasethe marking fluid being activatable to release the marking fluid suchthat the mixture of the nucleic acid marker and the marking fluid isdispersed onto the one or more bank notes.

Advantageously, the apparatus is an automated teller machine cassette.

Preferably the means to release the marking fluid comprises a reservoirof marking fluid.

Alternatively the marking fluid comprises an indelible ink.

Conveniently the nucleic acid marker is mixed in with the reservoir ofink.

Advantageously the marking apparatus further comprises a nucleic acidreservoir containing the nucleic acid marker, the nucleic acid reservoirbeing coupled to the distribution mechanism such that when the markingfluid reservoir is activated, the distribution mechanism mixes themarking fluid and the nucleic acid marker.

Alternatively the means to release a marking fluid comprises one or moresmoke pellets, the marking fluid comprising smoke.

Preferably the means to release the marking fluid and the distributionmechanism comprise a pyrotechnic device containing the one or more smokepellets.

Preferably the nucleic acid marker comprises a plurality ofsingle-stranded DNA oligonucleotides.

Advantageously each DNA oligonucleotide comprises a variable regionflanked by first and second generic regions on either side, there beingminimal homology between the variable region and the generic regions.

Conveniently each DNA oligonucleotide comprises a variable regionflanked by first and second generic regions, the variable regioncontaining no consecutively repeated nucleotides.

Preferably the marking apparatus further comprises a detector connectedto the means to release the marking fluid, the detector activating themeans to release the marking fluid in response to detecting tamperingwith the marking apparatus.

Advantageously the marking apparatus further comprises a plurality ofcarrier particles to which the nucleic acid marker is attached.

Conveniently the carrier particles are made from a polymer.

Preferably the carrier particles are made from an inorganic compound,more preferably magnesium silicate hydroxide.

Advantageously the nucleic acid marker is attached to the carrierparticles via a covalent bond.

Conveniently each nucleic acid marker molecule is bonded to a linkermolecule, the linker molecule having a covalent bond to a carrierparticle.

Preferably the linker molecule is a C₁₂-C₁₆ alkyl group.

Alternatively the nucleic acid marker is attached to the carrierparticles via ionic interactions or passive adsorption.

Preferably the marking apparatus further comprises a fluorescent labelattached to the carrier particles.

According to a second aspect of the present invention, there is provideda bank note storage system comprising a plurality of marking apparatusesas described above, wherein the nucleic acid markers each have arespective variable region flanked by first and second generic regions,the first and second generic regions being the same on all of thenucleic acid markers and the variable regions being different in thenucleic acid markers of each marking apparatus.

According to a third aspect of the present invention, there is provideda method of security marking an item comprising the steps of: providinga mixture of a nucleic acid marker and a marker fluid; and dispersingthe mixture onto the item.

Conveniently the method further comprises the step of identifying thenucleic acid marker.

Preferably the method further comprises the step of, prior to providinga mixture of the nucleic acid marker and the marker fluid, selecting thenucleic acid marker from a pool of nucleic acid markers, each marker inthe pool having a respective variable region flanked by first and secondgeneric regions, the first and second generic regions being the same onall of the nucleic acid markers in the pool and the variable regionsbeing different on each marker in the pool.

According to a fourth aspect of the present invention, there is provideda method of analysing an item that has been marked with a marker fluidand a nucleic acid marker, comprising the steps of: identifying thenucleic acid marker.

Conveniently, the step of identifying the nucleic acid marker comprisessequencing at least a portion of the nucleic acid marker.

Preferably, the step of identifying the nucleic marker comprises:providing a plurality of test oligonucleotides; applying the nucleicacid marker to the test oligonucleotides under conditions such that thenucleic acid marker hybridizes to test oligonucleotides to which it iscomplementary; and determining the test oligonucleotide oroligonucleotides to which the nucleic acid marker has hydrodised.

Advantageously, the test oligonucleotides are attached to a substrate inan array formation.

Preferably the item is a bank note, but is alternatively, one or morecredit cards, cellular phone recharging cards, tickets such as lotterytickets or confidential storable documents.

Advantageously the method further comprises the step of amplifying thenumber of copies of the nucleic acid marker sequence prior to the stepof sequencing at least a portion of the nucleic acid marker.

Conveniently the amplifying step comprises using thermal cycling nucleicacid amplification, preferably PCR, to amplify the number of copies ofthe nucleic acid marker sequence.

Alternatively, the amplifying step comprises using an isothermalamplification technique.

Preferably the method further comprises the step of measuring thequantity of amplified nucleic acid in the polymerase chain reactionduring the amplifying step and stopping the amplification after thequantity of amplified nucleic acid reaches a predetermined threshold.

Advantageously the step of measuring the quantity of amplified nucleicacid comprises the step of adding an intercalating dye to the amplifiednucleic acid.

Conveniently the nucleic acid marker comprises a plurality ofsingle-stranded DNA oligonucleotides.

Preferably each DNA oligonucleotide comprises a variable region flankedby first and second generic regions on each side, there being minimalhomology between the variable region and the generic regions.

Advantageously each DNA oligonucleotide comprises a variable regionflanked by first and second generic regions, the variable regioncontaining no consecutively repeated nucleotides.

Conveniently the method comprises using the marking apparatus describedabove.

Preferably, the method further comprises the step of removing thenucleic acid marker from the carrier particles.

Advantageously the method further comprises the step of visualising thefluorescent label and determining the location of the fluorescent labelon the item.

According to a fifth aspect of the present invention, there is provideda marking apparatus for marking an item, the apparatus comprising: apyrotechnic device containing one or more smoke pellets; a dye; and anucleic acid marker, the dye and nucleic acid marker being impregnatedinto the smoke pellets, the pyrotechnic device being activatable torelease a mixture of smoke, the dye and the nucleic acid marker.

According to a sixth aspect of the present invention, there is provideda method of producing a marking apparatus comprising: providing apyrotechnic device containing one or more smoke pellets having a dyeincorporated therein; preparing a mixture of a nucleic acid marker and asolvent; and positioning the mixture on to at least one of the smokepellets such that the mixture diffuses through the smoke pellets.

Preferably the mixture comprises between 60 and 90% alcohol and between10 and 40% water.

In certain aspects of the present invention, the step of measuring thequantity of amplified nucleic acid is used in methods of analysing anitem that has been marked with a nucleic acid marker but that has notbeen marked with a marker fluid.

It is to be understood that, in certain aspects of the presentinvention, the marking apparatus is not provided with means to receivethe item. In these aspects of the invention, the marking apparatus is aseparate module that can be attached to a suitable means to receive theitem.

In this specification a “marking fluid” means a fluid that visiblystains an item.

In this specification a “fluid” means any liquid or gas, includingsmoke, that has the properties of a fluid.

In this specification, the phrase “identifying a nucleic acid marker”means determining sufficient information about the marker such that itcan be differentiated from any other nucleic acid marker. In someembodiments this comprises sequencing the nucleic acid marker. Howeverin other embodiments sequencing, as such, does not take place becausethe nucleic acid marker is identified by, for example, determining thatit is identical to another nucleic acid marker.

In this specification, the word “comprising” means ”including” or“consisting of” and the word “comprises” means “includes” or “consistsof”.

In order that the present invention may be more readily understood, andso that further features thereof may be appreciated, embodiments of theinvention will now be described, by way of example, with reference tothe accompanying drawings in which:

FIG. 1 is a cross-sectional view of a bank note cassette in accordancewith one embodiment of the present invention;

FIG. 2 is a cross-sectional view of a bank note cassette in accordancewith another embodiment of the present invention;

FIG. 3 is a plan view, with part cut away, of a portion of a bank notecassette in accordance with a further embodiment of the invention; and

FIG. 4 is a schematic view of a DNA oligonucleotide attached to aparticle via a linker in accordance with a portion of a furtherembodiment of the invention.

Referring to FIG. 1, a bank note cassette 1 comprises an outer housing2, divided into a marking compartment 3 and a bank note storagecompartment 4. The bank note cassette 1 is of a type for insertion intoan ATM.

The marking compartment 3 contains a canister 5 of pressurised gasconnected via a pipe 6 and an actuator 7 to an ink reservoir 8. The inkreservoir 8 contains 200 ml of indelible ink mixed with 1 ml of DNA inbuffer, such that there is a total of between 5 and 80 nmoles of DNA inthe indelible ink.

The ink is such that it is difficult to resolublize or lead away. Insome embodiments the ink is an alcohol soluble dye with a pigment and asurface active agent in an organic solvent such as industrialisedmethylated spirits. An exemplary ink is disclosed in EP-A-0623658.

The DNA is added to the ink while the ink is in the ink reservoir 8, theink and DNA being mixed up and the reservoir 8 subsequently sealed. TheDNA is of a particular sequence which will be described in greaterdetail below.

A outlet pipe 9 leads from the ink reservoir 8, via a pressure valve 10to a distribution arm 11. The outlet pipe 9 passes into the bank notestorage compartment 4 of the housing 2 such that the distribution arm 11is located in the bank note storage compartment. The distribution arm 11is provided with numerous perforations (not shown) which allow the exitand dispersion of ink from the distribution arm 11. Located adjacent thedistribution arm 11, within the bank note storage compartment 4, are twostacks of bank notes 12 received in channels. A mechanism (not shown) isprovided to allow release of the bank notes 12 once the cassette 1 hasbeen inserted into an ATM.

In use, the contents of the marking compartment 3 and the distributionarm 11 are inactive while the cassette 1 is transported and the banknotes 12 are removed from an ATM under normal circumstances. However, ifthe cassette 1 is tampered with, a detection mechanism (not shown) sendsa signal to the actuator 7 to release the contents of the canister 5such that the pressurised gas forces itself into the ink reservoir 8.This raises the pressure within the ink reservoir 8 and the portion ofthe outlet pipe 9 leading up to the pressure valve 10. When the pressurein the ink in the pipe 9 reaches a predetermined level, the pressurevalve 10 releases itself and the ink and DNA mixture are forced out ofthe reservoir 8, through the remainder of the outlet pipe 9 and into thedistribution arm 11 where they are sprayed over the stacks of bank notes12. Thus the distribution arm 11 disperses the ink and DNA mixture. Thearm is located so that all of the bank notes 12 are covered with themixture of ink and DNA. Furthermore, the ink and DNA mixture isdispersed with such force that a portion of it leaves the bank notestorage compartment 4 and sprays around the exterior of the cassette 1.This will cover the individual who was tampering with the cassette withthe mixture of ink and DNA.

Thus, as soon as the cassette 1 is tampered with, each of the bank notes12 is covered not only in the indelible ink but also with the DNA.

In some variants of this embodiment, the ink reservoir 8 is collapsibleand the pipe 6 leading from the canister 5 to the ink reservoir 8 is notprovided. In these variants, the actuator 7 is situated on the canisterand, when activated, releases the contents of the canister into themarking compartment 3. This collapses the ink reservoir 8 and forces theink and DNA mixture out through the outlet pipe 9

It is to be understood that the cassette 1 is usually one of a pluralityof such cassettes which together form a bank note storage system. Whenbank notes need to be transported, one of the cassettes is selected,loaded with bank notes and then transported to an ATM in which it isinserted. When a cassette has been legitimately emptied of bank notes itis returned.

The DNA that is stored in the ink reservoir 8 will now be described ingreater detail. The DNA in the ink reservoir 8 comprises a plurality ofsingle-stranded oligonucleotides, each of which is identical. In a banknote storage system, in which a number of separate cassettes 1 areprovided, the oligonucleotides are provided with particular sequences.The oligonucleotides within each cassette 1 have the same sequence.However, comparing the oligonucleotide sequences of one cassette withthose of other cassettes in the bank note storage system, each cassettehas oligonucleotides with a variable central region such that eachcassette contains DNA having a distinct and identifiable oligonucleotidesequence. However, the variable region of each oligonucleotide isflanked by first and second generic regions. The first generic region onevery oligonucleotide in all the cassettes of the bank note storagesystem is the same and, similarly, the second generic region of theoligonucleotides in all of the cassettes of the bank note storage systemis the same. A database of the sequences of all of the variable regionsof the DNA markers and their respective cassette is maintained. Whenmanufacturing the cassettes of a bank note storage system, a pool ofsets of DNA oligonucleotides is provided, each set having a differentvariable region. A set of DNA oligonucleotides is selected from the pooland inserted into the ink reservoir 8 of one of the cassettes

As an example, the oligonucleotides in one of the cassettes of the banknote storage system has the sequence of SEQ ID NO:1 and theoligonucleotides in another of the cassettes has the sequence of SEQ IDNO:2. As can be seen, the sequences have central variable regions(underlined), which are different from one another, flanked by genericregions at their 5′ and 3′ ends which are the same in both sequences.The generic regions and the variable region are designed such that thereis minimal homology between the generic regions and the variable region.Minimal homology between two sequences is defined, in some embodimentsof the invention, as when two or, in other embodiments, more than twoconsecutive nucleotides in one sequence are not present consecutively inthe other sequence. 5′ACGTAGTAAAGAGGTGCCCGCCACTCGCTGTCGCAGATCATCGAGGGAAGACCACACGTGAGCCCAGAAC3′ SEQ ID NO:1 5′ACGTAGTAAAGAGGTGCCCGCCATGACATCGTCTGAGATCGAGCTGGAAGACCACACGTGAGCCCAGAAC3′ SEQ ID NO:2

Further explanation of these oligonucleotides is provided inWO-A-00/61799, which is incorporated herein by reference, in which theyare referred to as Type III tags.

After the bank notes 12 have been marked with the indelible ink and theDNA, and have been recovered, the bank notes 12 are analysed in order todetermine in which of the cassettes 1 they were originally stored. Inorder to analyse the marked bank notes, the DNA is extracted from thenotes using a solvent, which is then removed and any components in theDNA which would inhibit PCR are also cleaned from the DNA. The DNA isthen amplified using PCR. The primers used in order to amplify the DNAare complementary to the first and second generic regions of theoligonucleotides. Because there is no homology between generic regionsand the variable region, there can be no “false priming” during PCRamplification by primers erroneously binding to parts of the variableregion.

It is to be appreciated that, since the first and second generic regionsare the same for the oligonucleotides in all of the cassettes in thebank note storage system, the same primers are used in order to amplifythe oligonucleotide sequences no matter which cassette the bank notescome from in the storage system. Therefore, it is not necessary to knowin advance from which cassette the bank notes come in order to carry outPCR amplification of the oligonucleotide sequences.

Once the DNA oligonucleotides have been amplified to a sufficientquantity, they are sequenced using methods well known in the art.

However, it has been discovered that in the synthesis of DNAoligonucleotides using conventional methods, a particular sample ofmarker oligonucleotides having a certain sequence is often contaminatedwith a very small amount of contaminating oligonucleotides havingdifferent sequences. These other oligonucleotides are the remains ofprevious synthesis reactions. In practice, when oligonucleotides areprepared for insertion into the cassettes of a bank note storage system,it is often the case that the contaminating oligonucleotides in aparticular sample are the remains of oligonucleotides that weresynthesised for other cassettes.

Thus, when the ink and DNA mixture is dispersed onto bank notes, thebank notes are covered with a certain amount of the contaminatingoligonucleotides. Although the contaminating oligonucleotides are onlypresent in very small quantities, it is to be noted that they have thesame generic regions as the marker oligonucleotides and so they are alsoamplified in the PCR process. Because the initial quantity ofcontaminating oligonucleotides is much lower than that of the markeroligonucleotides, there is a time lag between significant amplificationof the marker oligonucleotides and significant amplification of thecontaminating oligonucleotides during PCR. Nevertheless, if the PCRprocess is allowed to run for too long, the quantity of contaminatingoligonucleotides becomes sufficiently high that the contaminatingsequences are also sequenced during the sequencing procedure. This leadsto confusing results where two or more sequences are determined for aparticular sample, either of which could be correct.

Therefore, in order to overcome this problem, in preferred embodimentsof the present invention, an intercalating dye such as SyBr Green™ isadded to the DNA sample during the PCR process. The intercalating dyebecomes visible under specific wavelengths of light in the presence ofdouble-stranded DNA. The quantity of double-stranded DNA in the reactionis calculated during the PCR process by measuring the level offluorescence of the intercalating dye under the illumination of light atthe excitation wavelength of the dye. This gives an indication of thequantity of amplified DNA in the sample while the PCR process takesplace. As soon as the quantity of amplified DNA has reached a level atwhich it can be sequenced, the PCR process is stopped so that thequantity of contaminating sequences in the sample does not reach a levelthat will affect the sequencing process. Thus only the nucleotidesequence of the marker oligonucleotides is determined.

By comparing the nucleotide sequence of the variable region that isdetermined against the database of the nucleotide sequences of the DNAin the cassettes in the bank note storage system, it is possible todetermine which of the cassettes the bank notes have come from. It istherefore known that the bank notes have come from the theft of thatparticular cassette and the owner of the bank notes can be determined.With this information, it is possible to arrange reimbursement of themarked bank notes.

Furthermore, because DNA is sprayed upon the individual who tamperedwith the cassette, the clothes, or even the skin, of the individual canbe analysed in a similar manner to the bank notes in order to establishthat the individual tampered with the cassette. This can subsequently beused as evidence in court.

It is to be appreciated that to set up a bank note storage system, adifferent nucleotide sequence is required in the variable region of theDNA for each cassette. Therefore, if the storage system comprises tenthousand separate cassettes (in, for example, a delocalised storagesystem), it is necessary for the variable region of the oligonucleotidesto be long enough such that it is able to encode at least ten thousandseparate sequences. Even so, the variable region of the oligonucleotidesneed not be particularly long. A variable region of twelve nucleotideswould be more than adequate for most purposes. However, if relativelyshort variable regions are used, particular sequencing methods areutilised in order to determine the sequence since traditional sequencingmethods may not be capable of accurately sequencing such a shortoligonucleotide.

For example, in some embodiments of the invention, Pyrosequencing™ isused. This technique is described in greater detail in WO-A-98/13523,WO-A-98/28440, WO-A-00/43540, EP-A-1141401 and WO-A-02/85341 which areincorporated herein by reference. In these embodiments, the variableregion of each of the oligonucleotides is synthesised such that eachnucleotide is always adjacent to a different type of nucleotide. Thus,for example, if the first nucleotide in the variable region is A thenthis may be followed by C, G or T, but not another A. This ensures thatthere are no consecutively repeated nucleotides thereby simplifying thesequence interpretation. The variability possible in the variable regionis somewhat reduced by this approach (only 3^(n) sequences are possibleinstead of 4^(n), where n is the length of the sequence) butoligonucleotides having sufficient length to encode the requisite numberof sequences can easily be synthesised.

As explained above, the generic regions and the variable region aredesigned such that there is minimal homology between the generic regionsand the variable region. This is achieved in these embodiments byincluding nucleotide repeats in the generic regions, which, as has beenexplained above, do not exist in the variable region.

In alternative embodiments of the invention, the DNA oligonucleotides donot comprise a central variable region flanked by two generic regions asdescribed above. In some of these alternative embodiments, the DNAoligonucleotides comprise a variable region adjacent a generic region(referred to as a Type I tag in the nomenclature of WO-A-00/61799). Inother embodiments, the DNA oligonucleotides comprise two adjacentvariable regions (referred to as a Type II tag in WO-A-00/61799). Inthese embodiments, the above described method of DNA amplification isadapted, as will now be described, to provide a plurality of differentprimers.

In DNA amplification relating to bank note storage systems wherein TypeI tags are used, a plurality of different primers, each complementary toone of the various variable regions and primers complementary to thegeneric region are provided. PCR amplification of the DNAoligonucleotide is then carried out with all of the primers present, toensure that amplification takes place no matter which variable regionsequence the oligonucleotide has. The amplified oligonucleotides arethen sequenced.

However, in a variation of this embodiment, the DNA oligonucleotides arefirst divided into a plurality of different samples. To each sample isadded primers complementary to one of the possible variable regions andprimers complementary to the generic region. The PCR process is thencarried out although this is only successful in the sample in which theprimers complementary to the variable region of the particular DNAoligonucleotide are present. In other samples, no complementary primeris present to prime the DNA polymerase and so DNA amplification does nottake place. By determining in which sample PCR amplification has beensuccessful and noting the particular variable region primer that wasused in that sample, it is possible to determine the sequence of the DNAoligonucleotide without a separate sequencing step.

In bank storage systems wherein Type II tags are used, PCR amplificationof the DNA oligonucleotides is carried out by adding a plurality ofprimers, each complementary to one of the various variable regions. PCRamplification of the DNA oligonucleotides is then carried out althoughonly the primers which are actually complementary to the variableregions of the DNA oligonucleotide function in the PCR process. Once DNAamplification has been completed, the DNA oligonucleotide is sequencedas described previously.

In a variation of this embodiment, the DNA oligonucleotides areinitially divided into a plurality of samples. Before the amplificationprocess, a different pair of primers is added to each sample, eachprimer being complementary to one of the possible variable regions ofthe DNA oligonucleotide. Amplification of the DNA oligonucleotides onlytakes place in that sample which is provided with primers which arecomplementary to the variable regions of the DNA oligonucleotide. Thus,by noting the sequence of the primers that were added to the samplewhich was amplified, it is possible to identify the particular DNAoligonucleotide without sequencing the oligonucleotide as such.

In some further embodiments of the present invention (in which Type I,Type II or Type III DNA oligonucleotides may be used) the DNAoligonucleotide is not identified by sequencing the oligonucleotide assuch. In these embodiments, a DNA oligonucleotide micro array is used toidentify the DNA oligonucleotide. Such DNA micro arrays are known in theart and are, for example, disclosed in EP-A-0373203, which isincorporated herein by reference.

The micro array comprises a substrate to which is attached a pluralityof single stranded test oligonucleotides of different sequences, in anarray. When it is necessary to identify a DNA oligonucleotide, from aDNA sample removed from a bank note, a label (such as a fluorescentlabel or a radioactive isotope) is attached to each DNA oligonucleotidein the sample. The sample of labelled DNA oligonucleotides is thenapplied to the substrate under conditions which are sufficientlystringent such that there is hybridization between the labelled DNAoligonucleotides and the test oligonucleotides attached to the substrateonly if the oligonucleotides are complementary to each other along theentire length of the test oligonucleotide. Non-hybridizedoligonucleotides are subsequently washed away. The position of thehybridized oligonucleotides is then determined by the location of thelabel on the array. By selecting the length and sequence of the testoligonucleotides carefully, it is possible for the pattern ofhybridizing oligonucleotides to identify exactly a particular sequence.By relating together the sequences of the test oligonucleotides thathybridized to the sample DNA oligonucleotide, it is possible todetermine the sequence of the sample DNA oligonucleotide.

However, even this step is unnecessary in certain embodiments in whicheach sequence of oligonucleotide in a bank note storage system isaccorded a security code which uniquely identifies the oligonucleotide.The security code is associated with a related database entry includinginformation such as the particular ATM cassette in which theoligonucleotide is stored. The pattern of the hybridizedoligonucleotides in the array is, as has previously been described,indicative of a particular oligonucleotide. A record is kept of eachpattern of hybridization and the security code of the correspondingoligonucleotide which forms that pattern of hybridization. When anoligonucleotide is analyzed on the micro array, a pattern ofhybridization is formed which is then compared with the record todetermine the security code and identify oligonucleotide. Thus, it ispossible to identify a particular DNA oligonucleotide marker withoutsequencing the oligonucleotide as such.

In the above described embodiments of the invention, the DNA is storedwith the ink in the reservoir 8 under normal circumstances. However, insome alternative embodiments of the invention, this is not the case.Referring to FIG. 2, a cassette 1 in accordance with such an alternativeembodiment is shown, in which like components are given the samenumbers. This embodiment of the invention is the same as the previousembodiments except that the reservoir 8 contains only the indelible inkand no DNA. Instead, a separate DNA reservoir 13 is provided, connectedto the pressure valve 10. The DNA reservoir 13 contains 1 ml of amixture of DNA in a buffer.

In use, this embodiment functions in the same way as the previousembodiment, and the contents of the marking compartment 3 do not operateunder normal circumstances. If the cassette 1 is tampered with then, aswith the previous embodiment, the compressed gas is released from thecanister 5 thus increasing the pressure in the reservoir 8. When thepressure in the reservoir 8 reaches a predetermined level, the pressurevalve 10 is released, allowing the simultaneous passage of ink throughthe outlet pipe 9 into the dispersing arm 11 together with the mixingtherein of the DNA contained in the second reservoir 13. Thus, in thisembodiment, a mixture of the ink 8 and the DNA is formed only when themarking mechanism is activated. This can be advantageous in embodimentsin which the ink degrades the DNA and also has the advantage that theparticular DNA stored in a cassette 1 can be changed without requiringthe reservoir of ink 8 also to be changed.

In some types of bank note cassettes, a marking mechanism other than anink marking mechanism is used. In particular, in some embodiments a“smoke and dye” marking mechanism is used instead. In these embodiments,the contents of the marking compartment 3 and the dispersing arm 11 arenot provided as has been described in the previous embodiments. Instead,one or more pyrotechnic devices are provided in the cassette 1. Suchpyrotechnic devices are known to those skilled in the art.

Referring to FIG. 3 a pyrotechnic device 14 comprises an outer housing15 in the centre of which is located a section of sand 16. An array ofsmoke pellets 17 is packed around the section of sand 16 within thehousing 15. The smoke pellets 17 have an indelible dye incorporated intothem.

FIG. 3 depicts part of the top of the housing 15 cut away so that theinterior of the pyrotechnic device 14 can be seen. However, as is shownat the bottom righthand corner of the device, the housing 15 covers allof the exterior of the device except for an aperture 18 that is providedin the housing of the pyrotechnic device 14. During manufacture, DNAoligonucleotides are added to the pyrotechnic device by dripping between200 μl and 1 ml of a solution of DNA in a mixture of 75% alcohol and 25%water through the aperture 18. On contact with the array of smokeparticles 17 beneath the aperture 18 the solutions diffuse across thearray until the DNA has soaked into each of the smoke pellets 17.

In use of this embodiment, the pyrotechnic device 14 is inactive undernormal circumstances, it being possible for bank notes 12 to be removedfrom the cassette 1 when they are dispensed properly while it is in anATM. However, if the housing of the cassette 1 is tampered with, adetector (not shown) sends a signal to the pyrotechnic device 14 whichactivates the pyrotechnic device. Upon activation, the smoke pellets 17undergo an exothermic reaction. The smoke pellets 17 very rapidlygenerate a large quantity of smoke, in which the dye and the DNAoligonucleotides are mixed. The smoke builds up inside the housing 15but can only escape through the aperture 18. Thus the pyrotechnic device14 not only acts as a means to release the smoke but also as adistribution mechanism for the smoke, dye and DNA mixture. Because thesmoke is a fluid and behaves accordingly and because it is generated insuch large quantities, it very quickly permeates throughout the interiorof the cassette 1 and comes into contact with each of the bank notes 12.The smoke carries the dye as well as the DNA and so each of the banknotes is marked with both the dye and the DNA.

Furthermore, some of the smoke is released from the cassette 1 and thiscovers the individual who tampered with the cassette with a mixture ofthe dye and DNA.

Following the marking of the bank notes in this way, the bank notes canbe analysed as is described in the previous embodiments. Similarly, theclothes, or even the skin, of the individual can be analysed aspreviously described.

In an alternative version of the “smoke and dye” embodiments of theinvention, the DNA oligonucleotides are not added to the pyrotechnicdevice 14 directly. Instead, the DNA oligonucleotides are first added toor incorporated within a plurality of micron or sub-micron sized carrierparticles. In some embodiments, these carrier particles are made frompolymers. In other embodiments, the carrier particles are inorganic incomposition, being made, for example, from talc (magnesium silicatehydroxide).

Referring to FIG. 4, a preferred embodiment of such a version is shownschematically, although only a portion of the surface of the particle isdepicted. Each DNA oligonucleotide 19 is bonded to a carrier particle 20by a linker molecule 21 such as a C₁₂-C₁₆ alkyl group. The linkermolecule 21 is bound, at one end 22, to the DNA oligonucleotide 19 and,at the other end 23, has a covalent bond with the particle 20. However,in some other embodiments, the DNA oligonucleotides are bound directlyto the particles through ionic interactions or by passive adsorption.

In particularly preferred embodiments which use carrier particles, afluorescent label 24 is also attached to each carrier particle 20.

The advantage of attaching the DNA oligonucleotides 19 to carrierparticles 20 is that it assists in the general identification of banknotes 12 which are marked with DNA oligonucleotides 19, especially whenthe carrier particles 20 are also marked with a fluorescent label 24.

In use, the bank notes 12 are first visualised (e.g. by subjecting themto ultraviolet light) in order to determine whether or not thefluorescent label 24 can be seen. If the fluorescent label is presentthen not only is the bank note 12 identified as being one which islabelled with the DNA oligonucleotide 19 but the particular area of thebank note 12 which has been so marked is also pinpointed. The steps ofDNA amplification and sequencing can then be carried out as previouslydescribed. Thus the use of a fluorescent label 24 on the carrierparticle 20 avoids the need for DNA amplification and sequencing ofevery bank note that is recovered, which would be unnecessary if thebank notes are ultimately found not to have been marked with a DNAoligonucleotide.

In the embodiments in which the DNA oligonucleotide 19 is attached tothe particle 20 via a linker molecule 21 of sufficient length (such asthe C₁₂-C₁₆ alkyl group described above) then steric hindrance of theDNA oligonucleotide 19 adjacent the particle 20 is avoided and the stepsof amplification and sequencing of the DNA oligonucleotide 19 can becarried out without removing each oligonucleotide 19 from its respectivecarrier particle 20.

In the embodiments in which the DNA oligonucleotide 19 is attacheddirectly to the particle 20 through an ionic interaction or by passiveadsorption then each DNA oligonucleotide 19 is first released from itsrespective particle 20 by putting the particles 20 and DNAoligonucleotides 19 in a buffer and changing the ionic strength of thebuffer, such as by changing its pH. Once each DNA oligonucleotide 19 hasbeen released from its particle 20, the amplification and sequencingsteps are carried out as previously described.

In the above described embodiments of the invention, amplification ofthe DNA oligonucleotides is carried out using the PCR process. However,in other embodiments of the invention, different amplification processesare used. For example, in some embodiments, a thermal cycling nucleicacid amplification technique other than PCR is used. In some otherembodiments of the invention, an isothermal amplification technique isused to amplify the number of copies of the DNA oligonucleotide.

It is to be appreciated that, while the above embodiments have beendescribed in relation to cassettes carrying bank notes, in alternativeembodiments the cassette 1 carries other, similar, valuable itemsinstead such as bank bonds or store vouchers.

It is to be understood that in certain variants of the aboveembodiments, the marking compartment 3 is a separate module that isdetachable from the bank note storage compartment 4. In these variants,a locking mechanism is provided between the marking compartment moduleand the bank note storage compartment 4 to lock the two compartmentstogether. Attempts to tamper with the locking mechanism will trigger therelease of the ink or smoke and dye and DNA mixture.

The above described embodiments of the invention relate to a cassettefor insertion into an ATM. However, in other embodiments of theinvention a different type of cassette is provided or even a differenttype of marking apparatus. For example, in one embodiment, the cassetteis of a type for the transfer of bank notes between banks and noprovision is made for insertion into an ATM. In another embodiment, themarking apparatus is a brief case which comprises a marking compartment3 and bank note storage compartment 4 substantially as described aboveexcept that the bank note storage compartment 4 is not adapted forinsertion into an ATM and is, instead, accessible from within the briefcase.

In a particularly preferred variant of the “smoke and dye” embodiment,instead of an ATM cassette, a money dye pack is provided. This pack isstored in the drawer of a human bank teller in a bank, and, in the eventof a robbery, is handed over to the thief. Instead of being activated bya tamper sensor, the release of the ink or smoke and dye and DNA mixtureis triggered by the pack leaving the confines of the bank.

In other respects, these embodiments work in a similar manner as thepreviously described embodiments.

1. A marking apparatus for marking an item, the apparatus comprising:means to receive the item; a nucleic acid marker; means to release amarking fluid; and a distribution mechanism coupled to the nucleic acidmarker and the means to release the marking fluid, the means to releasethe marking fluid being activatable to release the marking fluid suchthat the distribution mechanism disperses a mixture of the nucleic acidmarker and the marking fluid onto the item. 2-43. (canceled)
 44. Amarking apparatus according to claim 1 wherein the item is one or morebank notes, the means to release the marking fluid being activatable torelease the marking fluid such that the mixture of the nucleic acidmarker and the marking fluid is dispersed onto the one or more banknotes.
 45. A marking apparatus according to claim 44 wherein theapparatus is an automated teller machine cassette.
 46. A markingapparatus according to claim 1 wherein the means to release the markingfluid comprises a reservoir of marking fluid.
 47. A marking apparatusaccording to claim 46, wherein the marking fluid comprises an indelibleink.
 48. A marking apparatus according to claim 47, wherein the nucleicacid marker is mixed in with the reservoir of ink.
 49. A markingapparatus according to claim 46, further comprising a nucleic acidreservoir containing the nucleic acid marker, the nucleic acid reservoirbeing coupled to the distribution mechanism such that when the markingfluid reservoir is activated, the distribution mechanism mixesthe-marking fluid and the nucleic acid marker.
 50. A marking apparatusaccording to claim 1, wherein the means to release a marking fluidcomprises one or more smoke pellets, the marking fluid comprising smoke.51. A marking apparatus according to claim 50, wherein the means torelease the marking fluid and the distribution mechanism comprise apyrotechnic device containing the one or more smoke pellets.
 52. Amarking apparatus according to claim 1 wherein the nucleic acid markercomprises a plurality of single-stranded DNA oligonucleotides.
 53. Amarking apparatus according to claim 52, wherein each DNAoligonucleotide comprises a variable region flanked by first and secondgeneric regions on either side, there being minimal homology between thevariable region and the generic regions.
 54. A marking apparatusaccording to claim 52, wherein each DNA oligonucleotide comprises avariable region flanked by first and second generic regions, thevariable region containing no consecutively repeated nucleotides.
 55. Amarking apparatus according to claim 1 further comprising a detectorconnected to the means to release the marking fluid, the detectoractivating the means to release the marking fluid in response todetecting tampering with the marking apparatus.
 56. A marking apparatusaccording to claim 1 further comprising a plurality of carrier particlesto which the nucleic acid marker is attached.
 57. A marking apparatusaccording to claim 56 wherein the carrier particles are made from apolymer.
 58. A marking apparatus according to claim 56 wherein thecarrier particles are made from an inorganic compound, preferablymagnesium silicate hydroxide.
 59. A marking apparatus according to anyclaim 56 wherein the nucleic acid marker is attached to the carrierparticles via a covalent bond.
 60. A marking apparatus according toclaim 59 wherein each nucleic acid marker molecule is bonded to a linkermolecule, the linker molecule having a covalent bond to a carrierparticle.
 61. A marking apparatus according to claim 60 wherein thelinker molecule is a C₁₂-C₁₆ alkyl group.
 62. A marking apparatusaccording to claim 56 wherein the nucleic acid marker is attached to thecarrier particles via ionic interactions or passive adsorption.
 63. Amarking apparatus according to claim 56 further comprising a fluorescentlabel attached to each carrier particles.
 64. A bank note storage systemcomprising a plurality of marking apparatuses according to claim 1wherein the nucleic acid markers each have a respective variable regionflanked by first and second generic regions, the first and secondgeneric regions being the same on all of the nucleic acid markers andthe variable regions being different in the nucleic acid markers of eachmarking apparatus.
 65. A method of security marking an item comprisingthe steps of: providing a mixture of a nucleic acid marker and a visiblystaining indelible ink; and dispersing the mixture onto the item,wherein the item is a bank note or similar valuable item.
 66. A methodaccording to claim 65, further comprising the step of identifying thenucleic acid marker.
 67. A method according to claim 65, furthercomprising, prior to providing the mixture of the nucleic acid markerand the marker fluid, selecting the nucleic acid marker from a pool ofnucleic acid markers, each marker in the pool having a respectivevariable region flanked by first and second generic regions, the firstand second generic regions being the same on all of the nucleic acidmarkers in the pool and the variable regions being different on eachmarker in the pool.
 68. A method of analysing an item that has beenmarked with a visibly staining indelible ink and a nucleic acid marker,comprising the steps of: identifying the nucleic acid marker; whereinthe item is a bank note or similar valuable item.
 69. A method accordingto claim 66 or 68 wherein the step of identifying the nucleic acidmarker comprises sequencing at least a portion of the nucleic acidmarker.
 70. A method according to claim 66 or 68 wherein the step ofidentifying the nucleic acid marker comprises: providing a plurality oftest oligonucleotides; applying the nucleic acid marker to the testoligonucleotides under conditions such that the nucleic acid markerhybridizes to test oligonucleotides to which it is complementary; anddetermining the test oligonucleotide or oligonucleotides to which thenucleic acid marker has hybridized.
 71. A method according to claim 70wherein the test oligonucleotides are attached to a substrate in anarray formation.
 72. A method according to claim 65 or 68 furthercomprising the step of amplifying the number of copies of the nucleicacid marker sequence prior to the step of identifying the nucleic acidmarker.
 73. A method according to claim 72, wherein the amplifying stepcomprises using thermal cycling nucleic acid amplification to amplifythe number of copies of the nucleic acid marker sequence.
 74. A methodaccording to claim 73, wherein the thermal cycling nucleic acidamplification is PCR.
 75. A method according to claim 72, wherein theamplifying step comprises using an isothermal amplification technique.76. A method according to claim 73, further comprising the step ofmeasuring the quantity of amplified nucleic acid in the polymerase chainreaction during the amplifying step and stopping the amplification afterthe quantity of amplified nucleic acid reaches a predeterminedthreshold.
 77. A method according to claim 76, wherein the step ofmeasuring the quantity of amplified nucleic acid comprises the step ofadding an intercalating dye to the amplified nucleic acid.
 78. A methodaccording to claims 65 or 68, wherein the nucleic acid marker comprisesa plurality of single-stranded DNA oligonucleotides.
 79. A methodaccording to claim 78, wherein each DNA oligonucleotide comprises avariable region flanked by first and second generic regions on eitherside, there being minimal homology between the variable region and thegeneric regions.
 80. A method according to claim 77, wherein each DNAoligonucleotide comprises a variable region flanked by first and secondgeneric regions, the variable region containing no consecutivelyrepeated nucleotides.
 81. A method according to claim 65 or 68comprising using the marking apparatus of claim
 1. 82. A methodaccording to claim 65 or 68 comprising using the marking apparatus ofclaim 20 and further comprising the step of removing the nucleic acidmarker from the carrier particles.
 83. A method according to claim 65 or68 comprising using the marking apparatus of claim 21, and furthercomprising the step of visualising the fluorescent label and determiningthe location of the fluorescent label on the item.
 84. A markingapparatus for marking an item, the apparatus comprising: a pyrotechnicdevice containing one or more smoke pellets; a dye; and a nucleic acidmarker, the dye and nucleic acid marker being impregnated into the smokepellets, the pyrotechnic device being activatable to release a mixtureof smoke, the dye and the nucleic acid marker.
 85. A method of producinga marking apparatus comprising: providing a pyrotechnic devicecontaining one or more smoke pellets having a dye incorporated therein;preparing a mixture of a nucleic acid marker and a solvent; andpositioning the mixture on to at least one of the smoke pellets suchthat the mixture diffuses through the smoke pellets.
 86. A methodaccording to claim 85 wherein the mixture comprises between 60 and 90%alcohol and between 10 and 40% water.